Separation of spiramycins by countercurrent distribution



s 011 947 SEPARATIGN F SPKRAMYCINS BY COUNTER- CURRENT DISTRBRUTION 3,011,947 Patented Dec. 5, 1961 "ice the form of tablets with potassium bromide. Position of the absorption bands expressed in cm.-'

Jean Preudhornrne and Yvan Charpenti, Paris, France, 5

assrgnors to Societe des Usines Chimiques Rhone- F I V H 3356 m Poulenc, Paris, France, a French body corporate i V L i x w No Drawing. Filed Jan. 12, 1956, Ser. No. 558,609 ggg 3,398 s- 2,338 sg s rong. I s rong. s tong. Claims priority, application France Nov. 30, 1955 2,940 strong 2,940 strong. 2,940 strong. 2 Claims. (Cl. 167-65) 1,735 strong. 1,740 strong. 1,740str0r1g;

1,455 medium. 1,457 medium. 1,460 medium.

The present invention relates to new antibiotics, salts 1373 medium Elme'hum' thereof, and to processes for the preparation thereof. 7 1,372 medium. 1,310 medium.

A new antibiotic splramycin is described in the publica lfi 1 300 P .1300

medium. me rum. tron Antibiotics Annual 1954-1955, pages 724-727, 1,275 medium 1,275 medium 17280 medium published February 20, 1955, by Med1cal Encyclopedia 1,237 medium 1,240 e i Inc., New York City. This antibiotic is produced by wsmmg' 1185 medium fermentation of the Streptomyces strain 8-3486 (NRRL a 1,160 strong. 1,160 strong. 1,162 strong. No. 2420), to which there has been g1ven the name 1,522 strong. 1,122 strong. 1,132 strong.

w e- 085 1 2 m;- amycm' T mammal a mnfture of 1,052 very strong. 1,052 very strong. 1,052 very strong. different constituents of very s1m1lar chemical compos1- LOB-strong; 1,015 strong. 1,015 strong. tion and as stated in the said application the existence of 993 smug gig figg fifi Strong these three consjltugmts' was analytically revealed y 905 medium. tram-aim. 906medium. counter-current o1str1but1on by means of a Craig ap- 86 feeble. @medium. sssroebio.

840 medium. 840 medium. 842 medium; i using. iyclohexane an aqueous splramycm 8l0feeble. 8l0fecble. 810 medium. solution contaimng 1% of dlSOdlllm phosphate. The use 782 medium. 782 medium. 782 medium. of cyclohexane however as difficult owing to the relative- 685 medium ly low solubility of spiramycin in that solvent. The present invention has for its principal object the provi- I sion of processes for the separation from the spiramycin p v V p complex of the individual spiramycin constituents which, The Spectral dlfierences between thfeeiproducts for convenience are identified herein as spirarnycin I, spiramycin I1 and spiramycin 111.

The physical and biological characteristics of these spiramycin constituents are as follows:

TABLE I are principally differences in the relative intensity of the absorption bands. These differences are found for the spectra in solution in carbon tetrachloride, which shows that they are not due to different stat%' of crystallisation.

Spiramycln I Spiramycin II Spirarnycin III Crude fOfmllla 4a-1sHnss u-17N: C4u'4aH10-az01s10Nz M lB WES Hfl S L Elementary Composition:

0, percent 60.3 H, per 1 8.7.-. 0, percent. 7 28.5.- N, percent 3.2 Molecular weight (ehullioscopy) about 800 Neutral equiv t pKli 7.7 76 M1. (on he Mariuenne block) 134-137" C [odmu' (c.=1%, methanol) 96 [a]D2o (c 1%, ethanol) 91 [11]]32 (c.=1%, chloroform) 57 Ultraviolet spectrum (in solution in ethanol):

W'avelength of the maximum absorption 232 mu E11; at 232 IIEp a w; Chromatography on paper 1 Rf L 0.04 0.15 0.22;

1 Chromatography on Whatman paper No. 1 impregnated with hufitr having a; pH valre 9 minim, 12320 at a concentrathan 23.8 g.,l.) using as developer solvent the light phase of the cycbl e caneiehylisobutyl e-tor e-water system (85:15:25 volumes) Descending technique-displacement of the solvent front cm. in 4 hours at 25 0. Biological developmenton sewn gelose plate.

In Table II, the main infra-red absorption bands for the three products are indicated:

TABLE II Infra-red absorption spectrum of spiramycin bases I, I1 and III The spectrum was measured on the solid product in The individual constituents of spirarnycinare therefore defined as follows:

Spiramycin I.-A basic substance soluble in Chlori na'ted solvents, alcohols, hexane, aromatic hydrocarbons, keton'es, ethyl acetate and amyl acetate and capable of forming salts with acids, which basic substance contains the elements carbon, hydrogen, oxygen and nitrogen in substantially the following proportions by weight:

which basic substance displays, in ethyl alcohol solution a maximum absorption at 232 III/L, has a molecular weight determined by the ebullioscopic method of about 800, a neutral equivalent of 463, a dissociation constant pKb of 7.7, a melting point on the Maquenne block of 134- 137 C., has optical rotations [a] in methanol (c.=l%) of 96, in ethanol (c.=l%) of -91 and in chloroform (c.=l%) of 57, and which in solid form exhibits characteristic absorption in the infra-red region of the spectrum at the following frequencies expressed in reciprocal centimetres: 3470, 2970, 2940, 1735, 1455, 1378, 1317, 1275, 1237, 1160, 1122, 1090, 1052, 1015, 993, 905, 865, 840, 810, 782.

Spiramycin I1.--A basic substance soluble in chlorinated solvents, alcohols, hexane, aromatic hydrocarbons, ketones, ethyl acetate and amyl acetate and capable of forming salts with acids, which basic substance contains the elements carbon, hydrogen, oxygen and nitrogen in substantially the following proportions by weight:

which basic substance displays, in ethyl alcohol solution a maximum absorption at 232 m has a molecular weight determined by the ebullioscopic method of about 800, a neutral equivalent of 464, a dissociation constant pK of 7.6, a melting point on the Maquenne block of 130-133" C., has optical rotations [111 in methanol (c.=l%) of -86, in ethanol (c.=l%) of -80 and in chloroform (c.=l%) of -55, and which in solid form exhibits characteristic-absorption in the infra-red region of, the spectrum at the following frequencies expressed in reciprocal centimetres: 3460, 2970, 2940, 1740, 1457, 1372, 1300, 1275, 1232, 1160, 1122, 1085, 1052, 1015, 993, 940, 905, 860, 840, 810, 782, 685

' Spimmycin 1II.-A basic substance soluble in chlorinated solvents, alcohols, hexane, aromatic hydrocarbons, ketones, ethyl acetate and amyl acetate and capable of forming salts with acids, which basic substance contains the elements carbon, hydrogen, oxygen and nitrogen in substantially the following proportions by weight:

Carbon 61 Hydrogen 8.5 Oxygen 26.7 Nitrogen 3.0

which basic substance displays, in ethyl alcohol solution a maximum absorption at 232 my, has a molecular weight determined by the ebullioscopic method of about 900, a neutral equivalent of 473, a dissociation constant pKb of 7.6, a melting point on the Maquenne block of 128-131 M., has optical rotations [11:1 in methanol (c.=l%) of 83, in ethanol (c.=l%) of -79 and in chloroform (c.=1% of 50, and which in solid form exhibits characteristic absorption in the infra-red region of the spectrum at the following frequencies expressed in reciprocal method of fractional crystallisation and the method of chromatographic separation.

According to one embodiment of the present invention a process for the separation of the aforesaid constituents from spiramycin comprises subjecting a solution of spiramycin to treatment in a counter-current distribution apparatus employing as phases an organic phase consisting of an aromatic hydrocarbon or chlorinated hydrocarbon solvent, or mixture thereof, and an aqueous phase consisting of a salt solution buffered to pH 6 to 7, the two phases being initially mutually saturated, isolating the constituents from the organic phase fractions after rendering those fractions alkaline and evaporating the solvent and in the case of constituents II and III further purifying said constituents by crystallisation from an aromatic hydrocarbon solvent.

Separation by counter-current distribution may be carried out, for example, in a Craig apparatus (A. Weissberger, Technique of Organic Chemistry, Interscience Publishers, New York, vol. III, p. 286). It is possible to work at a temperature between 0 and 50 C., but it is preferable to work at about 2025 C. There is employed, in accordance with the present invention, a solvent of the aromatic hydrocarbon group (such as ben zene) or a chlorinated solvent (such as dichloroethane) or mixtures of these solvents. There is used as the second phase a buffer solution having a pH value between 6 and 7. The two phases are previously mutually saturated, that is to say, there is used for the separation, for example, the two phases separated by decanting a mixture composed of one volume of the organic solvent and one volume of the aqueous phase.

The spiramycin to be treated is brought into solution in a fraction of the organic phase. This fraction is equal to the volume of the organic phase to be introduced into one or more of the first'cells of the counter-current distribution apparatus. It would be ideal to be' able to introduce all the spiramycin into the first cell, because in this case a better separation would be effected, but a limit is set by questions of solubility if it is desired to treat relatively large quantities of product. An acceptable separation is eifected by distributing the spiramycin in the first two, three or four cells.

The fractionation is effected in the manner usual in this type of separation, and the various constituents are isolated from the fractions containing them by extraction in the organic phase (after making alkaline), and thereafter evaporating the solvent from this phase. In the case of spiramycins II and III this is followed by crystallisation from a solvent of the aromatic hydrocarbon group, such for example as benzene.

According to a further embodiment of the invention a process for the separation of the said constituents from spiramycin comprises subjecting a solution of spiramycin to chromatography on alumina. If it is desired to separate spiramycin I from spiramycins II and III, it is advantageous to use a good solvent for spiramycins, such as an aromatic hydrocarbon (for example benzene) or ether. In this case, spiramycins II and III are separated from spiramycin I by elution by this same solvent. The spiramycin I remains on'the column and is thereafter washed out by a benzene-acetone or benzene-alcohol mixture or by alcohol or acetone.

For separating the spiramycin II from the spiramycin III, a less eifective solvent for spiramycins is employed, such as a mixture of cyclohexane and an aromatic hydrocarbon or a mixture of cyclohexane and ether. The elution by this same mixture gives fractions enriched in spiramycin III, while the spiramycin 11 remains on the column and can be washed out by ether and then by a mixture of ether and acetone.

On repeating these chromatographic operations, spiramycins I, II and III are obtained in the pure state. The bases are isolated from the fractions containing them by evaporation of the organic solvent, followed in the case of spiramycins II and III by crystallisation from an aromatic hydrocarbon solvent, such for example as benzene.

According to a further embodiment of the invention a process for the separation of the said constituents from spiramycin comprises subjecting spiramycin to fractional crystallisation from an aromatic hydrocarbon solvent, preferably benzene. It is possible by this method to separate the mixture of bases II and III, which crystallises from benzene, from base I, which remains in the mother liquor. The amorphous base I is then purified either by counter-current distribution or by chromatography with alumina. Bases II and III can be separated either by counter-current distribution or by chromatography with alumina, followed by recrystallisation from benzene.

Spirarnycins I, H and III have substantially the same anti-bacterial spectrum and the same activity as the starting spirarnycin.

The following examples show by way of non-limitative example how the invention can be carried into practice.

EXAMPLE I 20 g. of spiramycin in the form of its crude base are fractionated in a Craig apparatus comprising 60 cells, each having a capacity of 200 cc. The solvents employed consist of the two separate phases of the following mixture:

Benzene 1 volume. Buffer solution pH 6.47 (KH PO 6.35 g., Na i-IP 121-1 0: 7.14 g.; water to make 1 litre) Do.

100 cc. of aqueous phase are introduced into each of the cells of the Craig apparatus disposed in a chamber at 24 C. and the spiramycin is dissolved in 200 cc. of the benzene phase. The distribution is then eifected by the usual method. 100 cc. of spirarnycin solution in the henzene phase are introduced into the first cell, shaken and decanted, and the benzene phase is transferred into the second cell; a further 100 cc. of spiramycin solution in the benzene phase is then introduced in the first cell. After agitation and decanting, the benzene phase is transferred from the second cell into the third cell, and that from the first cell into the second cell. The fractionation is continued in this way, introducing 100 cc. of benzene phase into the first cell after each decanting operation.

When 59 transfers in series have been effected, the separation is ended. The contents of each cell are collected, the aqueous phase is made alkaline at pH 9-10 by means of dilute caustic soda, agitated and the benzene phase is decanted. A second extraction of the aqueous phase is effected by means of 40 cc. of benzene, which is combined with the first decanted phase. The total volume of the benzene extract is adjusted to 150 cc. The dry extract is determined on an aliquot part of each benzene solution, and a curve is plotted to represent the concentration of dry extract in each cell. Three concentration maxima are obtained, which correspond to the following partition ratios of the three constituents:

Cell having the maximum concentration Partitlon rati0= Gone. in benzene phase/Cone. in aqueous phase Product A Cells to 15. Product B Cells 21 to 28. Product C Cells 35 to 45.

The benzene solutions containing a common constituent in the pure state are collected, the solvent is driven oil by U distillation under reduced pressure and the distillation residue is dried in vacuo (6 hours at 40 C. under 1 mm.).

There are thus obtained:

G. Product A 7.8 Product B 2.9 Product C 3.4

Product A is dried to constant weight in a vacuum oven (40 C. 1 mm. for 12 hours).

It has the following characteristics:

These constants are those of spiramycin I (Table I).

- Chromatography on Whatrnan paper No. 1 impregnated with bufier solution (Na HPO 12H O at a concentration of 23.8 g./l. at pH 9), with the light phase of the cyclohexane-methylisobutylketone-water system 15:25 volumes) as developer solution, using the descending technique with a 4-hour development at 25 C., shows after bioautography on a nutrient agar plate inoculated with B. subtilis the presence of a single product characterised by an R of 0.04.

2 g. of spiraniycin I base are converted into the corresponding sulphate by dissolution of the base in 5 cc. of methanol and adjusting the pH value of the solution to 5.5 by the addition of a dilute sulphuric acid solution in methanol. The salt is precipitated by adding the methanol solution to 250 cc. of anhydrous ether with agitation. The precipitate is centrifuged and dried.

Weight of sulphate obtained:1.95 g. S0 percent:10.0 113 (c.=2%, water) :73i1

It is also possible to prepare other salts, such as the hydrochloride, the nitrate, the perchlorate and the phthalate by the same method.

2 g. of product B are dissolved in 4 cc. of benzene at boiling temperature, the solution is cooled to 10 C. and after 15 hours the crystals obtained are centrifuged, washed with 0.2 cc. of benzene and dried. 1.57 g. of crystallised product melting at 129-132 C. is thus obtained.

A second crystallisation carried out under the same conditions gives a product having a melting point of 130- 133 C. and having the following characteristics:

Composition: C%=61.6; H%=8.5; O%=26.8; N%

[M (c.=l%, methanol)=86 ltraviolet spectrum (in solution in ethanol):maximum absorption at 232 mp.

( li's.

These constants are those of spiramycin II (Table I).

Chromatography on paper effected under the same conditions as for spiramycin I indicates the presence of a single product characterised by an Rf of 0.15.

2 g. of spiramycin II base are converted into sulphate.

Weight of sulphate obtained: 1.9 g. 80;, percent:9.8 1 (c.=2%, water) :72il

3 g. of the product C are dissolved in 6 cc. of benzene at boiling temperature, the solution is cooled to 10 C. and after 15 hours the crystals obtained are centrifuged, washed with 0.3 cc. of benzene and dried. 1.9 g. of crystallised product melting at l27l30 C. is thus obtained.

A second crystallisation carried out under the same conditions gives a product having a melting point of 128-131 C. having the following characteristics:

Composition: C%=61; H%=8.5; %=26.7; N%=3.0 4 (c.=1%, methanol)- =83 8 The relative compositions were determined by chromatography on paper under the conditions described above.

Fractions 7, 8, 9 and 10 are collected and concentrated under reduced pressure and the base obtained is UISII'DZVIOIEt spect2r;12mm(1n solution 1n ethanol) .rnaxrmum dried in vacuo O for 20 hours at 1 mm).

Orp on a F I The base thus prepared consists of spiramycin I having (E:t...=327) the following characteristics: These constants are those of spiramycin III (Table I). Melting point on the Maquenne block: 1334350 0 Chromatography on paper carried out under the same [a]D2o(c =1% methanol): conditions as for spirarnycin I indicates the presence of a Ultrawiolet Spectrum (in Solution of ethanol) single product characterised by a Rf of 0.22. l

2 g. of spiramycin 111 base are converted into sulphate. 7 12"... at 3 i 316 We-oht of Sul hate obtainedzLfi Chromatography carried out on paper under the consoicpercemzgj g 15 ditions of Example I shows that the product contains a [(111320 (c =2% 0 0 single constituent characterised by an Rf of 0.04.

g. of spiramycin base II+III obtained by three E.XAMPLE H successive crystallisations and having the following com- 100 g. of spiramycin in the form of its crude base are position; dissolved at boiling temperature in lZQ cc of benzene, the 20 Percent by weight solution is cooled to +10 C. and maintained under slow Spiramycin U a 56 agitation for 15 hours, and the crystals obtained are cens an 4 trifuged, washed with cc. of benzene and dried, thus constituting the fi t f ti are fractionated 1n the Craig apparatus used in the course The mother liquors, to which the washing liquors are 9 h sephfahoh describe? 111 E p The p added, are concentrated in vacuo to 60 cc. and the soluhon lshalrled out as descflhed 111 h eXamPk With the tion obtained is subjected to repeated crystallisation by followlhg System Of Solvents! the same method whereby a second fraction is obtained. B

enzene 1 volume. a 2 $222 obtamed by evaporating the mother 30 Buffer solution having a pH value of 6.24

50 g. of the first fraction are recrystallised from 100 g zg iz'fi g Eig 12H2O:4'8 D0 cc. of benzene as hereinbefore indicated, and the crystals obtained are washed with 20 cc. 0f 1361126116 and dried. when the distribution has been completed, the aque- 29 g. of crystallised product are obtained. ous phases are rendered alkaline and extracted with A ihll'd cl'ystalhsahoh 20 of the above p f h benzene and the dry extract of each benzene solution is from Q benzene Elves 14 of f l comahhhg determined. The distribution curve shows two maxima 3 p y 11 and the Sharacterlstlcs of Which corresponding to spiramycins II and III characterised are as follows! i by the following partition ratios: Spiramycin II content-56% by weight 40 I Spiramycin III content-44% by Weight Cellsd having Partitionbratio= MP. (on the Maquenne block)-l93-140 C. I 2 60m in [u] (c.=1%methanol)86 mmmm 5.1 5333%;

The spiramycin H and III contents, just given were 5 determined by chromatography on paper under the conggig'gg ditions defined in Example I.

25 g. of base enriched with spiramycin I by crystallisation of the mixture of II and III and emanating from the ,Compamon the experimental dlsmbutlon cuive third fraction of the first crystallisation are dissolved in with the curve calculated from the values of the partition 125 cc. f ether and the solution is chromatographed on ratio found show that the two constituents are in the a column f alumina (wgight f alumina; 500 diam pure state in the solutions emanating from the following eter of the columnz28 mm.). After the passage of the cells: ethereal solution the chromatograph is developed with Sir am cln II Cells6to 15. ether, the ob ect of which is to eliminate spiramycms II g g 111" Cells 2110 3L and HI. Spiramycin'I is then washed out bypassing a mixture of ether and acetone (1:1 by volume) through the column.

The progress of the chromatography is summarised in the following table:

The benzene solutions corresponding to these two groups of cells are dried and the bases obtained are crystallised from benzene as described in Example I.

7.2. g. of spiramycin *II and 5.1 g. of spiramycin III IMAM in crystallised form are thus obtained, the characteristics of which are as follows:

are dissolved in 450 cc. of a mixture of cyclohcxanc and ether (2:1 by volume) and the solution is chromatographed on an alumina column (weight of alumina: 600

Spiramycin ll iSpiramycin HT Characteristics of the products obtained:

Spiramyoin II Spiramycin III MI. on the Maquenne block 130132 C 128-130 C. [alu (c.=1%1nethunol) 5 -82. Ultra-violet spectrum (in solution in ethanol):

12%,, at 232 m, 305 325. Chromatography on paper a single product--. a single product. Chromatography on paper Rf 0.15 0.22.

EXAMPLE IV 50 g. of spiramycin in the form of its crude base are dissolved in 500 cc. of benzene and the solution is chromatographed on a column containing 750 g. of alumina (diameter of the column: 40 mm.). After the passage of the benzene solution, the chromatograph is developed with benzene, which has the effect of Washing out all the spirarnycins II and III. A mixture of benzene and acetone (1:3 by volume) is thereafter used for the development, which has the effect of Washing out the spiramycin I.

The progress of the chromatography is summarised by the following table:

g., a diameter of the columnz35 mm.). The chromatograph is developed first by the same mixture of cyclohexane and ether and then successively by ether and by a mixture of ether and acetone (1:1 by volume).

The progress of the chromatography is summarised by the following table:

The relative compositions were determined by chromatography on paper under the conditions described above.

Fraction 3 is concentrated under reduced pressure and the residue is dried and thereafter crystallised from 12 cc. of benzene as indicated in Example I. 4 g. of spiramycin III in the form of its crystallised base are thus Obtained.

The treatment of fraction 12 under the same conditions, followed by a crystallisation by the method indicated in Example I, gives 5.8 g. of crystallised spiramycin II in the form of its base.

Fractions 6, 7 and 8 are collected and concentrated under reduced pressure and the base obtained is dried in a vacuum oven (40 C. for 20 hours at 1 mm.). The base thus prepared consists of spiramycin I having the following properties:

Melting point on the Maquenne block: 133-l34 C. [111 (c.=1% methanol): 94 C. Ultra-violet spectrum (in solution in ethanol) Elfi at 232 mp: 318

Chromatography on paper carried out under the conditions of Example I shows that this base contains a single constituent characterised by an R of 0.04.

By further chromatography of the mixture of bases of combined fractions 1 and 2, in accordance with the method indicated in Example III, it is possible to obtain bases II and III in the pure state in the form of crystallised products.

We claim:

1. Process for obtaining separately spiramycin I, spiramycin II and spiramycin III which comprises subjecting a crude material containing the same to countercurrent distribution in a Craig countercurrent distribution apparatus of 60 cells, effecting 59 transfers at a temperature of about 20-25 C. with an organic phase consisting of benzene and an aqueous phase bufiered at a hydrogen-ion concentration within the range pH 6 to pH 7, the two phases being initially mutually saturated, isolating the constituents from the organic phase fractions after rendering those fractions alkaline and evaporating the solvent, the spiramycin I being in cells 5 to 15, spirarnycin II being in cells 21 to 28 and spiramycin III being in cells 35 to 45, and in the case of spiramycins II and III subjecting the products to fractional crystallisation by dis- 1 1 l 2 solving them in a hot aromatic hydrocarbon solvent and Swart: J. Clin. Inv., September 1949, vol. 28, pp. allowing the resulting solution to cool. 10451046.- 7

2. Process as defined in claim 1 wherein the aromatic Newton et 21.: Biochem. 1., pp. 597-603, vol. 53, No. hydrocarbon solvent is benzene. 4, March 1953.

5 Zechmeister: Progress in Chromatography, 1938-1947, References Cited in the file of this Patent pp. 247-260, pub. 1950, by John Wiley and Sons, Inc.

UNITED STATES PATENTS Baron Handbook of Antibiotics, pub. by Reinhold Pub.

v Co., N.Y.C., 1950, pp. 265-266. 3:223:33 EifiilQiiiiiiiiiI: $23? 5%, 13?? AntibioticsAnnual 195A-1955pn72fl-727and 10 830, pub. February 1955, by Med. Encyclopedia Inc. OTHER REFERENCES Craig et 211.: J. Biol. Chem, -vol. 16, 1945, pp. 321-332.

Leach et al.: JACS, vol. 69, 1947, p. 474. 

1. PROCESS FOR OBTAINING SEPARATELY SPIRAMYCIN I, SPIRAMYCIN II AND SPIRAMYCIN III WHICH COMPRISES SUBJECTING A CRUDE MATERIAL CONTAINING THE SAME TO COUNTERCURRENT DISTRIBUTION IN A CRAIG COUNTERCURRENT DISTRIBUTION APPARATUS OF 60 CELLS, EFFECTING 59 TRANSFERS AT A TEMPERATURE OF ABOUT 20-25*C. WITH AN ORGANIC PHASE CONSISTING OF BENZENE AND AN AQUEOUS PHASE BUFFERED AT A HYDROGEN-ION CONCENTRATION WITHIN THE RANGE PH 6 TO PH 7, THE TWO PHASES BEING INITIALLY MUTUALLY SATURATED, ISOLATING THE CONSTITUENTS FROM THE ORGANIC PHASE FRACTIONS AFTER RENDERING THOSE FRACTIONS ALKALINE AND EVAPORATING THE SOLVENT, THE SPIRAMYCIN I BEING IN CELLS 5 TO 15, SPIRAMYCIN II BEING IN CELLS 21 TO 28 AND SPIRAMYCIN III BEING IN CELLS 35 TO 45, AND IN THE CASE OF SPIRAMYCINS II AND III SUBJECTING THE PRODUCTS TO FRACTIONAL CRYSTALLISATION BY DISSOLVING THEM IN A HOT AROMATIC HYDROCARBON SOLVENT AND ALLOWING THE RESULTING SOLUTION TO COOL. 